Purdue University biologist Christie Sahley and research assistant Orie Shafer have
found that nitric oxide synthase, or NOS, is activated when parts of the nerve cell
are damaged in the medicinal leech, a three-inch-long invertebrate known for its
ability to regenerate its neural connections.

The Purdue group now is conducting follow-up studies to see what role NOS may play
in neural regeneration. The findings may someday be applied to research in human
spinal regeneration. Details of the study were presented Nov. 21 at the annual meeting
of the Society for Neuroscience.

"Our study shows that NOS in the leech is activated at the site of injury within minutes
after axons are severed, and it remains active well beyond 48 hours after the injury,"
Sahley says. Axons are the long "arms" of a nerve cell that carry impulses away from the cell body toward a target cell.

"We are now conducting follow-up studies to identify what information this molecule
provides at a cellular level, and to see how these functions might help the leech's
nervous system set itself up to allow regeneration to occur," she says.

NOS, an enzyme also found in humans, produces a "signaling" molecule that sends chemical
messages throughout the body to incite certain chemical reactions. Because the NOS
found in leeches is very much like the human NOS, it may serve a similar function
in both species, Sahley says.

"Nerve cells in leeches are basically identical in structure and function to those
in more complicated systems such as humans," Sahley says. "But for some reason, nerve
regeneration in higher systems is not complete. By analyzing how nerves regenerate
in a simple system, we may find clues to facilitate regeneration in vertebrates."

When activated, NOS produces nitric oxide, a gas that in recent years has been recognized
to act as a signaling molecule in vertebrates, including humans. As a signaling molecule,
nitric oxide sends chemical messages to other molecules, setting up a chain of command to produce a specific response or set off a series of chemicals reactions.

Studies show that the NOS enzyme plays a role in a number of functions, such as learning
and feeding, and is activated in response to neuronal injury in several organisms,
including mammals.

Though it has been tied to a number of functions, scientists have not yet identified
how NOS operates at a cellular level.

"We suspect that NOS plays an important role in the organization of many events, but
we have not yet determined how it sends its signals or what specific molecules it
targets," Sahley says. "Our goal in this study is to narrow it down and see, in a
regenerating system, who this protein talks to and what it does at any specific time."

Her studies suggest that nitric oxide may serve as a "stop sign" for microglia, specialized
cells that roam the central nervous system to ingest foreign particles, such as bacteria
and dead cells. By flagging down microglia to help at the site of injury, nitric oxide may aid efforts to minimize tissue damage and infection. Sahley is pursuing
studies to test this hypothesis.

Sahley says the medicinal leech, Hirudo medicinalis
, is an ideal model for studying the basic processes behind neural regeneration. "Not
only do their neurons reconnect after injury, but they have been shown capable of
making the right connections to fully restore motor function," she says.

Humans and other vertebrates, by comparison, have only limited capacity to restore
neural growth or function.

One problem with studying neural regeneration in vertebrate systems is that there
are hundreds of thousands of neurons, making it impossible to identify what each
individual cell does, she says.

By contrast, the medicinal leech has 21 identical columns of neurons, called ganglia,
which run up and down its body. Each ganglion is made up of only 400 neurons.

"Because we're looking at the connections between 400 neurons, rather than thousands,
we can track the NOS to an individual cell and monitor its function, both within
the cell and within the system," she says.

The 21 ganglia that make up the nervous system of the leech "talk" to each other through
a connective, a structure somewhat like a spinal cord without the spine. The connective
is made up of numerous axons that allow the ganglia to send messages to each other.

In her study, Sahley and Shafer used a pair of fine forceps to crush the connective,
severing the axons running from ganglia to ganglia. The injury was immediately followed
by a staining process to detect the presence of active NOS.

Their findings showed that the NOS remained localized to the site of the injury --
never staining beyond the pinch -- indicating that NOS plays an active role at the
site where the injury occurs.

"We found one particular form of NOS that shows up only at the site of the crush,"
Sahley says. "We are now conducting follow-up studies to identify what information
this particular signaling molecule gives the regenerating nervous system."

In addition, the staining results suggest the nitric oxide might be involved in the
rapid microglial accumulation that follows injuries to the connectives.

Sahley and Shafer cultured some of the pinched connectives in the presence of an NOS
inhibitor to see what happened when the signaling process was blocked. After 24 hours
of regeneration, the groups without the inhibitor showed tight gatherings of microglia around the area of injury, while those treated with the inhibitor showed a scattered
distribution of microglia, spread well beyond the site of injury.

Sahley now has several experiments under way to test whether NOS acts as a "stop sign"
to hold microglia at the site of injury. She also is working to identify other molecules
within cells that may play a role in the regeneration process.

The research is funded by the National Institutes of Health and the National Institute
of Mental Health.

The medicinal leech, once used in medicine to draw blood from patients, may provide
new clues to neural regeneration. Purdue researcher Christie Sahley and her research
assistant, Orie Shafer (background) are using the leeches to analyze the role nitric
oxide synthase may play in neural regeneration. (Purdue News Service Photo by David
Umberger)

Activation of Nitric Oxide Synthase (NOS) in response to neuronal injury has been
detected in several organisms. We nave investigated the role of active NOS in the
regenerating nervous system of the medicinal leech Hirudo Medicinalis. Through the
use of NADPH diaphorase staining, an indicator of active NOS, we have shown that this protein
is activated within minutes of injury to segmental connectives. The injury, delivered
by a pinch with fine forceps, was immediately followed by intense staining for NADPH
diaphorase activity, indicating the presence of active NOS. Sites of injury stained
positively for active NOS well beyond 48 hours after in vivo pinches were delivered
to segmental connectives. Furthermore, the staining remained localized to the site
of injury, never staining beyond the pinch. Following crushes to leech segmental connectives,
microglia actively migrate to the site of injury. Our staining results suggested
that Nitric Oxide (NO) might be involved in the rapid microglial accumulation that
follows injuries to connectives in the medicinal leech. Culturing pinched connectives
either in the presence of the NOS inhibitor L-nitro-arginine-methyl-esther (L-NAME),
or its inactive isomer D- NAME showed that NO may serve as a stop signal for migrating
microglia. For short regeneration times (2 to 6 hours) the distributions of microglia
about the sites of lesions were virtually indistinguishable. However, after 24 hours
of regeneration D-NAME-treated chords, just like untreated chords, showed tight aggregates of microglia within the area of injury while L-NAME-treated chords displayed
a diffuse unaggregated distribution spread well beyond the site of injury. Our results
suggest that NO serves as signal for microglia to stop at the sites of injury resulting in the accumulation and aggregation of these cells at the sites of regenerating axon
outgrowth. Supported by NS34927 (CLS).